CAJ | 학술논문

为建立菜用大黄最佳的SRAP反应体系，进一步对菜用大黄品种进行遗传多样性分析，以菜用大黄基因组DNA为模板，采用单因素与正交设计相结合的方法，对SRAP-PCR扩增体系中的模板DNA、Mg2＋、dNTPs、引物和Taq DNA聚合酶浓度5个因素进行优化，并确立菜用大黄SRAP-PCR的最佳体系。结果表明：菜用大黄各因素对反应体系影响大小依次为：引物浓度＞Mg2＋浓度＞dNTPs浓度＞Taq DNA聚合酶浓度＞DNA用量；最佳反应体系为：25μL反应体系中含10×PCR Buffer 2．50μL、模板DNA 30 ng、Mg2＋2．50 mmol/L、dNTPs 0．25 mmol/L、Taq DNA聚合酶0．04 U/μL、上下游引物各0．5μmol/L。用8个菜用大黄品种DNA样品对优化的反应体系进行验证，均获得了条带清晰且多态性较好的扩增图谱，证实了该体系的稳定性和可靠性，可用于菜用大黄的遗传多样性分析。
위건립채용대황최가적SRAP반응체계，진일보대채용대황품충진행유전다양성분석，이채용대황기인조DNA위모판，채용단인소여정교설계상결합적방법，대SRAP-PCR확증체계중적모판DNA、Mg2＋、dNTPs、인물화Taq DNA취합매농도5개인소진행우화，병학립채용대황SRAP-PCR적최가체계。결과표명：채용대황각인소대반응체계영향대소의차위：인물농도＞Mg2＋농도＞dNTPs농도＞Taq DNA취합매농도＞DNA용량；최가반응체계위：25μL반응체계중함10×PCR Buffer 2．50μL、모판DNA 30 ng、Mg2＋2．50 mmol/L、dNTPs 0．25 mmol/L、Taq DNA취합매0．04 U/μL、상하유인물각0．5μmol/L。용8개채용대황품충DNA양품대우화적반응체계진행험증，균획득료조대청석차다태성교호적확증도보，증실료해체계적은정성화가고성，가용우채용대황적유전다양성분석。
The purpose of this study was to determine the best system of SRAP reaction and provide a founda -tion for further studying the genetic diversity of culinary rhubarb .Using the genome DNA as template ,and combi-ning the single-factor experiment with the orthogonal design , five factors including the concentrations of template DNA,Mg2+,dNTPs,Taq DNA polymerase and primers in SRAP-PCR reaction were optimized ,and then the optimal SRAP-PCR reaction system for culinary rhubarb was established .The results indicated that the order of each factor affecting the result of PCR was:primer>Mg2+>dNTPs>Taq DNA polymerase dosage>DNA.The optimal SRAP-PCR reaction for culinary rhubarb in a 25μL reaction system was 10 ×PCR Buffer 2.50 μL,template DNA 30 ng, Mg2+2.50 mmol/L,dNTPs 0.25 mmol/L, Taq DNA polymerase dosage 0.04 U/μL and each primer 0.5μmol/L. The optimized system was verified by the genomic DNA samples from eight cultivars of culinary rhubarb , and the clear bands and abundant polymorphism were obtained .It was concluded that the SRAP-PCR reaction system was stable and reliable ,and was suitable to analyze the genetic diversity of culinary rhubarb .